title: Engineering flavonoid glycosyltransferases for enhanced catalytic efficiency and extended sugar-donor selectivity.
abstract: Flavonoids are predominantly found as glycosides in plants. The glycosylation of flavonoids is mediated by uridine diphosphate-dependent glycosyltransferases (UGT). UGTs attach various sugars, including arabinose, glucose, galactose, xylose, and glucuronic acid, to flavonoid aglycones. Two UGTs isolated from Arabidopsis thaliana, AtUGT78D2 and AtUGT78D3, showed 89 % amino acid sequence similarity (75 % amino acid sequence identity) and both attached a sugar to the 3-hydroxyl group of flavonols using a UDP-sugar. The two enzymes used UDP-glucose and UDP-arabinose, respectively, and AtUGT78D2 was approximately 90-fold more efficient than AtUGT78D3 when judged by the k(cat)/K(m) value. Domain exchanges between AtUGT78D2 and AtUGT78D3 were carried out to find UGTs with better catalytic efficiency for UDP-arabinose and exhibiting dual sugar selectivity. Among 19 fusion proteins examined, three showed dual sugar selectivity, and one fusion protein had better catalytic efficiency for UDP-arabinose compared with AtUGT78D3. Using molecular modeling, the changes in enzymatic properties in the chimeric proteins were elucidated. To the best of our knowledge, this is the first report on the construction of fusion proteins with expanded sugar-donor range and enhanced catalytic efficiencies for sugar donors.
authors.affiliation: Department of Bioscience and Biotechnology, Bio/Molecular Informatics Center, Konkuk University, Seoul, 143-701, Korea.
authors.forename: Hye Soo
authors.initials: HS
authors.lastname: Kim
authors.forename: Bong-Gyu
authors.initials: BG
authors.lastname: Kim
authors.forename: Suhyun
authors.initials: S
authors.lastname: Sung
authors.forename: Mihyang
authors.initials: M
authors.lastname: Kim
authors.forename: Hyejung
authors.initials: H
authors.lastname: Mok
authors.forename: Youhoon
authors.initials: Y
authors.lastname: Chong
authors.forename: Joong-Hoon
authors.initials: JH
authors.lastname: Ahn
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics*; D029681:Arabidopsis Proteins / Q000235:genetics*; D020134:Catalytic Domain / Q000235:genetics; D016695:Glycosyltransferases / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011993:Recombinant Fusion Proteins / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D014532:Uridine Diphosphate Glucose / Q000235:genetics / Q000378:metabolism*; D014539:Uridine Diphosphate Sugars / Q000235:genetics / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D011993:Recombinant Fusion Proteins; D014539:Uridine Diphosphate Sugars; C040817:uridine diphosphate arabinose; D016695:Glycosyltransferases; D014532:Uridine Diphosphate Glucose